Allergic diseases, including nasal allergy, asthma, urticaria and angioedema, are among the most common diseases encountered by physicians in their clinical practice. Allergy refers to certain diseases in which a wide spectrum of biologically active substances, released from activated mast cells, cause tissue inflammation and organ dysfunction. In essence, any allergic reaction may lead to tissue damage in one or more target organs (see for example Lichtenstein, 1993).
On the cellular level, mast cells are significant mediators of the allergic reaction and are packed with 500 to 1000 granules in which the mediators of the inflammatory reactions are stored. These include vasoactive mediators such as histamine, chemotactic mediators and proteolytic enzymes. In addition, following the activation of mast cells, a number of mediators are generated de novo and released. These include arachidonic acid metabolites such as leukotrienes and prostaglandins and a number of multifunctional cytokines. Mast cell derived factors also recruit and activate additional inflammatory cells, such as eosinophils, neutrophils and mononuclear cells. Therefore, mast cell derived mediators possess all the requisite properties to induce the symptoms of itching, swelling, coughing and choking that are associated with an allergic reaction (Bienenstock et al., 1987). These mediators are released in response to processes which occur through a number of different pathways within mast cells. Thus, therapeutic treatments for allergy and related inflammatory conditions must intervene at some point in the allergenic pathway in order to be effective.
Current therapies against allergy include H1 and H2 blockers, which block the biological activities of histamine. Examples include chlorpheniramine, azatidine, ketotifen, loratidine and others. However, anti-histamines cannot counteract the inflammatory reactions effected by the additional mediators released alongside histamine. Therefore, anti-histamines cannot provide a reliable protection against allergy.
A better allergy treatment would block the secretory process by preventing mast cell degranulation. Drugs which are currently available for this purpose include hydrocortisone and disodium cromoglycate. However, disodium cromoglycate cannot inhibit all types of histamine secretion, and is not always completely effective. Steroids, on the other hand, are effective for blocking mast cell degranulation, but have many unacceptable side effects. Therefore, therapeutic agents which could prevent mast cell degranulation without significant side effects, and could thus prevent or significantly reduce the occurrence of clinical symptoms associated with allergy, such as neurogenic inflammation (see below for details), would be very useful for the treatment of allergy and related conditions.
Mast cell degranulation is a complex process involving at least two different pathways. Mast cells secrete their granular contents in a process of regulated exocytosis (degranulation) by two major pathways, the IgE (immunoglobulin E) dependent pathway and the IgE independent pathway. The IgE dependent pathway is invoked in response to an immunological trigger, brought about by aggregation of the high affinity receptors (Fc∈RI) for IgE, which are present on the cell surface of mast cells. This response involves crosslinking of cell bound IgE antibodies by the corresponding antigens (allergens).
The IgE-independent or peptidergic pathway is invoked in response to a number of polycationic compounds, collectively known as the basic secretagogues of mast cells. These compounds include the synthetic compound 48/80, naturally occurring polyamines and positively charged peptides, such as the neurotransmitter substance P (Ennis et al., 1980; Sagi-Eisenberg 1993; Chahdi et al., 1998).
The ability of substance P to induce mast cell degranulation, together with the observed presence of mast cells clustered around nerve endings which contain substance P, implicate mast cells as the mediators of substance-P induced neurogenic inflammation (Foreman 1987a,b; Pearce et al., 1989). It is well established that in the skin and elsewhere neurogenic inflammation, through the release of neurotransmitters such as substance P, is a contributor to a variety of diseases such as acute urticaria, psychogenic asthma, interstitial cystitis, bowel diseases, migraines, multiple sclerosis and more (Reviewed by Theoharides 1996). In addition, this IgE independent pathway of degranulation can also be evoked by snake, bee and wasp venoms, bacterial toxins and certain drugs such as opiates.
Although the signal transduction pathways by which mast cell degranulation is activated are not yet fully resolved, a number of cellular events have been shown to occur after stimulation of the mast cells. These include activation of phospholipases such as PLC, PLD and PLA2, elevation of cytosolic Ca2+ and activation of serine and tyrosine kinases (reviewed by Sagi-Eisenberg, R. “Signal Transmission Pathways in Mast Cell Exocytosis”. In: The Handbook of Immunopharmacology. Academic Press, UK. pp. 71-88, 1993).
Within these processes, however, the involvement of GTP-binding proteins (G-proteins) is well established. For example, the introduction of nonhydrolyzable analogues of GTP, such as GTP-γ-S, into ATP−4 permeabilized mast cells, stimulates PLC activity and degranulation.
From these and other observations, the involvement of at least two different G-proteins, one involved in PLC and Ca2+ activation (GP) and one directly regulating exocytosis (GE), has been suggested (Gomperts 1990; Gomperts et al., 1991; reviewed by Sagi-Eisenberg 1993). Indeed, it was subsequently demonstrated that basic secretagogues induce histamine secretion by interacting directly with GE, a pertussis toxin-sensitive heterotrimeric G protein, in a receptor-independent manner (Aridor et al., 1990; Aridor & Sagi-Eisenberg 1990). This G-protein was subsequently identified as Gi3, which appears to mediate the peptidergic pathway leading to exocytosis in mast cells. In particular, a synthetic peptide which corresponds to the C terminal sequence of Gαi3 (KNNLKECGLY (SEQ ID NO:1)) was able to inhibit histamine release when introduced, into permeabilized mast cells (Aridor et al., 1993).
However, the cell membrane is generally impermeable to most peptides. Therefore, the use of a peptide as a therapeutic agent, directed against an intracellular target, requires a special mechanism to enable the peptide to overcome the membrane permeability barrier.
One possible approach is based on the fusion of the selected peptide with a specific hydrophobic sequence, comprising the “h” region of a signal peptide sequence. Examples of such hydrophobic regions are the signal sequence of the Kaposi fibroblast growth factor (AAVALLPAVLLALLAP (SEQ ID NO:27); Lin et al., 1995; Rojas et al., 1997) and the signal sequence within human integrin β3 (VTVLALGALAGVGVG (SEQ ID NO:28); Liu et al., 1996; Review by Hawiger 1997).
Specific importation of biologically active molecules into cells by linking an importation-competent signal peptide to the molecule of interest was disclosed in U.S. Pat. No. 5,807,746, although only in vitro studies were described, such that the signal peptide was not shown to function in vivo. The signal peptide causes the entire complex to be imported into the cell, where theoretically the biologically active molecule could then have its effect. Although such direct importation could serve to introduce the therapeutic compound into the cell, the efficacy of the complex may be limited, such that the biologically active molecule may have little or no effect. The variables which may affect the efficacy of the biologically active molecule include the effect of linking the molecule to the signal peptide, which may result in an inactive hybrid molecule; unpredictable effects of the entire complex within the cell; and even the inability of the entire complex to be imported into the cell, despite the presence of the signal peptide.
In addition, identifying a suitable biologically active molecule for treatment of allergy may also be difficult. For example, linking a non-peptide molecule, such as a known secretion-blocking compound, to a signal peptide is both difficult and may result in an unstable molecule. A peptide could be used as the secretion-blocking compound, but then such a peptide must be carefully selected and tested. Finally, the entire complex would require testing, particularly in vivo, since the ability to penetrate a cell in tissue culture does not necessarily predict the efficacy of the complex in a human or animal subject. U.S. Pat. No. 5,807,746 therefore suffers from the drawback that only in vitro data is disclosed, such that the effect of the signaling peptides in vivo, alone or as part of a complex, is not known. Thus, suitable, targeted, specific therapeutic agents for the treatment of allergy are not currently available and are potentially complex and difficult to develop.
There is therefore a need for, and it would be useful to have, a therapeutic agent for the treatment of allergy and related inflammatory conditions, which would block mast cell degranulation and hence the release of histamine, but which would be specifically targeted to the degranulation pathway and which would therefore have few side effects.
Previous work has demonstrated the ability of several peptides to block mast cell degranulation. For example, a novel peptide designated Peptide 2, that was designed and synthesized to include an importation competent signal peptide, as a first segment at the N-terminus (underlined), and the C-terminal sequence of Gαi3 as a second segment at the C-terminus (AAVALLPAVLLALLAPKNNLKECGLY, SEQ ID NO:23) inhibited histamine release from activated mast cells (WO 00/78346). Additional active peptides in that disclosure include: 
The present invention is not intended to encompass any of the peptides disclosed and claimed in that application, and they are specifically excluded from the present invention, as are any known peptides according to the principles disclosed hereinbelow.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically indicated to be incorporated herein by reference. In addition, citation of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.